Christin Lorenz (Presenter)
Leibniz-Institut für Analytische Wissenschaften
Authorship: Sebastian Malchow (1), Christina Looße (1), Albert Sickmann (1), Christin Lorenz (1)
(1) Leibniz-Institut für Analytische Wissenschaften – ISAS – e.V., Bunsen-Kirchhoff-Straße 11, Dortmund, Deutschland
Platelets are primarily known to be key players in thrombosis and hemostasis. Targeting platelet function and signaling may represent novel therapeutic strategies in prevention of cardiovascular diseases. The absolute quantification of phosphorylation sites in actived platelets can direct the establishment of new diagnostic assays by characterization of samples with clinical relevance, including quantification of platelet receptors and signaling proteins and their regulation via posttranslational modifications in human blood. Therefore, this study aims to establish a targeted mass spectrometry based assay for quantitative analysis of the phosphorylation status in activated platelets, in particular ADP receptors and the related cAMP/PKA signalling cascade proteins.
Cardiovascular disease (CVD) is the leading cause of death globally, accounting for about 31% of all deaths. Platelets are primarily known to be key players in thrombosis and hemostasis and contribute to the genesis and progression of cardiovascular diseases. A variety of factors, including collagen, fibrinogen, adenosine diphosphate (ADP), von Willebrand factor (VWF), thrombin, thromboxane and others, promote platelet adhesion and aggregation by utilizing multiple intracellular signal transduction mechanisms. In addition to activating factors, platelets circulating in vivo are also continually exposed to inhibitory factors such as endothelium-derived nitric oxide (NO) and prostacyclin (PGI2). Most of these activating and inhibiting factors bind to specific platelet receptors to stimulate distinct signaling pathways, which promote or inhibit platelet adhesion, aggregation, and secretion (Jurk and Kehrel, 2005). A well-regulated equilibrium between these two opposing processes is assumed to be essential for normal platelet and vascular function. Consequently, impairment of this equilibrium will promote thrombotic, inflammatory or bleeding disorders. Owing to their anucleate nature, platelets have limited protein synthesis. Therefore, dynamic changes of post-translational modifications (PTM) are an important module of signal transduction during platelet activation and inhibition. The overall protein activity and regulation determine platelet function. The platelet proteins are derived from the megakaryocyte and the plasma and are adjusted after platelet formation due to external stimuli (Schwertz et al. 2010). Our previous work defined the platelet proteome composed of more than 5000 proteins, including novel receptors and signaling proteins (Burkhart et al. 2012). Some of these are already used as targets in antiplatelet therapy. One well-known example is the inhibition of cyclo-oxygenase (COX)-1 by the drug aspirin (Bhatt et al. 2006). Due to the increasing amount of information on platelet function in thrombotic diseases (Sexton and Smyth 2014) and platelet disorders (D´Andrea et al. 2009) precise diagnosis and therapeutically monitoring are feasible. One can assume that many functional disorders of platelets are related to changed levels of protein expression and altered PTM pattern.
In detail, platelets are prepared from fresh blood donations and activated respectively inhibited. The platelet activation is induced by addition of ADP and inhibited by Iloprost, a stable prostacyclin analogue, followed by quantification of the post-translation modification pattern of the ADP receptors and related proteins of the cAMP/PKA signaling cascade. Per sample, 100 mg of platelets will be digest and phosphopeptides enriched using TiO2, followed by a targeted mass spectrometry (LC-MS/MS) analysis on QExactive HF (Thermo Scientific) using heavy labeled phosphopeptides.
ADP is secreted by activated platelets representing a very important amplification mechanism to recruit additional platelets to sites of vascular injury. ADP and ATP are also released into blood vessels by dying vascular cells (Gachet 2008). In detail, ADP binds to the G-protein coupled membrane receptors P2Y1 and P2Y12 followed by stimulation of intracellular signaling cascades. The activation of the 7-transmembrane domain receptor P2Y1 stimulates calcium mobilization, platelet shape change, and rapid and reversible platelet aggregation and stimulation of the P2Y12 receptor coupled to a Gi-protein enhances amplification of stable platelet aggregation and secretion. P2Y12 inhibitors are among the most successful antiplatelet drugs, however, show remarkable variability in efficacy. Recently, we applied quantitative temporal phosphoproteomics to study ADP-mediated signaling at unprecedented molecular resolution and provide temporal profiles of 4797 phosphopeptides, 608 of which showed significant regulation (Beck et al. 2017). This data demonstrated that ADP-triggered phosphorylation occurs predominantly within the first 10 seconds, with many short rather than sustained changes, demonstrating an extensive spectrum of human platelet protein phosphorylation in response to ADP, which inversely overlap and represent major activating pathways. Targeting those PTM pattern may represent novel therapeutic strategies in prevention of cardiovascular diseases.
Conclusions & Discussion
This study aims to establish a targeted mass spectrometry based assay for quantitative analysis of the phosphorylation status in activated platelets, in particular ADP receptors and related signalling cascade proteins.The absolute quantification of protein phosphorylation allows the characterization of potential marker proteins representing an activation state of platelets associated with disease mechanisms. Thus, supporting the development of more sensitive and more selective mass spectrometry based diagnostic tests.
References & Acknowledgements:
Beck F., Geiger J., Gambaryan S., et al. Temporal quantitative phosphoproteomics of ADP stimulation reveals novel central nodes in platelet activation and inhibition. Blood. 2017;129(2):e1-e12. doi:10.1182/blood-2016-05-714048.
Bhatt D.L., Steg P.G., Ohman E.M., Hirsch A.T., Ikeda Y., Mas J.L., et al. International prevalence, recognition, and treatment of cardiovascular risk factors in outpatients with atherothrombosis. JAMA 2006 295:180-189.
Burkhart J.M., Vaudel M., Gambaryan S., Radau S., Walter U., Martens L., Geiger J., Sickmann A., Zahedi R.P. The first comprehensive and quantitative analysis of human platelet protein composition allows the comparative analysis of structural and functional pathways. Blood 2012 120:e73-e82.
D’Andrea G., Chetta M., Margaglione M. (2009) Inherited platelet disorders: thrombocytopenias and thrombocytopathies. Blood Transfusion 7(4):278-292.
Gachet C. P2 receptors, platelet function and pharmacological implications. Thromb Haemost. 2008;99(3):466-472.
Jurk K., Kehrel B.E. Platelets: physiology and biochemistry. Semin Thromb Hemost. 2005 31:381-392.
Schwertz H., Köster S., Kahr W.H.A., et al. Anucleate platelets generate progeny. Blood 2010 115(18):3801-3809.
Sexton T., Smyth S.S. Novel mediators and biomarkers of thrombosis. Journal of thrombosis and thrombolysis 2014 37(1):1-3.
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